Marine environments are a rich source of Actinobacteria and have the potential to produce a wide variety of biologically active secondary metabolites. In this study, we used four selective isolation media to culture Actinobacteria from the sediments collected from the Chukchi Shelf in the Arctic Ocean. A total of 73 actinobacterial strains were isolated. Based on repetitive DNA fingerprinting analysis, we selected 30 representatives for partial characterization according to their phylogenetic diversity, antimicrobial activities and secondary-metabolite biosynthesis genes. Results from the 16S rRNA gene sequence analysis indicated that the 30 strains could be sorted into 18 phylotypes belonging to 14 different genera: Agrococcus, Arsenicicoccus, Arthrobacter, Brevibacterium, Citricoccus, Janibacter, Kocuria, Microbacterium, Microlunatus, Nocardioides, Nocardiopsis, Saccharopolyspora, Salinibacterium and Streptomyces. To our knowledge, this paper is the first report on the isolation of Microlunatus genus members from marine habitats. Of the 30 isolates, 11 strains exhibited antibacterial and/or antifungal activity, seven of which have activities against Bacillus subtilis and Candida albicans. All 30 strains have at least two biosynthetic genes, one-third of which possess more than four biosynthetic genes. This study demonstrates the significant diversity of Actinobacteria in the Chukchi Shelf sediment and their potential for producing biologically active compounds and novel material for genetic manipulation or combinatorial biosynthesis.